CB2 expression in mouse brain: from mapping to regulation in microglia under inflammatory conditions.

Since its detection in the brain, the cannabinoid receptor type 2 (CB2) has been considered a promising therapeutic target for various neurological and psychiatric disorders. However, precise brain mapping of its expression is still lacking. Using magnetic cell sorting, calibrated RT-qPCR and single-nucleus RNAseq, we show that CB2 is expressed at a low level in all brain regions studied, mainly by few microglial cells, and by neurons in an even lower proportion. Upon lipopolysaccharide stimulation, modeling neuroinflammation in non-sterile conditions, we demonstrate that the inflammatory response is associated with a transient reduction in CB2 mRNA levels in brain tissue, particularly in microglial cells. This result, confirmed in the BV2 microglial cell line, contrasts with the positive correlation observed between CB2 mRNA levels and the inflammatory response upon stimulation by interferon-gamma, modeling neuroinflammation in sterile condition. Discrete brain CB2 expression might thus be up- or down-regulated depending on the inflammatory context.

Electroacupuncture-induced cannabinoid receptor expression in repair of abducens nerve.

Objective: Through the development of beagle abducens nerve injury model, taking electroacupuncture as the core and microglia as the starting point, the author investigated whether electroacupuncture can promote the repair of injured abducens nerve by cannabinoid receptor-mediated regulation of microglia activation. Methods: Healthy beagle dogs were randomly divided into five groups: sham operation group (A), injury group (B), electroacupuncture pretreatment group (C), antagonist group (D) and solvent group (E). After stimulation with electroacupuncture, the expression of cannabinoid 1 receptor (CB1R) and cannabinoid 2 receptor (CB2R) in A, B and C microglia cells was detected by Western Bolt analysis, and further the expression of CB2R in five groups was further analyzed by immunofluorescence, thereby statistical differences were analyzed. Results: Among group A, group B and group C, Western Blot analysis showed that there were no significant changes in the expression of CB1R protein after electroacupuncture [F (2, 12)=1.75, p = 0.215]. After electroacupuncture preconditioning for 15 min for 2 weeks, group C was compared with group A and group B, which showed CB2 was affected. The expression of CB2R protein was significantly increased among groups A, B and C [F (2, 12)=5189.57, p < 0.001], but there was no significant difference in the expression of CB2R protein between group A and group B (p > 0.05). The results of immunofluorescence showed that Arginse/CD11b was significantly increased in group C comparing to group A (*p < 0.001), while there was a significant increase in group E comparing to group A about Arginse/CD11b [F (4, 20)=4345.44, p < 0.001]. Conclusions: The CB2R in the cannabinoid receptor is mainly involved in the electro-acupuncture-induced neuroprotection. Electroacupuncture can promote the repair of injured abducens nerve by CB2R-mediated activation of microglia.

Selective Photoaffinity Probe That Enables Assessment of Cannabinoid CB2 Receptor Expression and Ligand Engagement in Human Cells.

Chemical tools and methods that report on G protein-coupled receptor (GPCR) expression levels and receptor occupancy by small molecules are highly desirable. We report the development of LEI121 as a photoreactive probe to study the type 2 cannabinoid receptor (CB2R), a promising GPCR to treat tissue injury and inflammatory diseases. LEI121 is the first CB2R-selective bifunctional probe that covalently captures CB2R upon photoactivation. An incorporated alkyne serves as ligation handle for the introduction of reporter groups. LEI121 enables target engagement studies and visualization of endogenously expressed CB2R in HL-60 as well as primary human immune cells using flow cytometry. Our findings show that strategically functionalized probes allow monitoring of endogenous GPCR expression and engagement in human cells using tandem photoclick chemistry and hold promise as biomarkers in translational drug discovery.

In vivo TSPO and cannabinoid receptor type 2 availability early in post-stroke neuroinflammation in rats: a positron emission tomography study.

BACKGROUND: Upregulated levels of 18-kDa translocator proteins (TSPO) and type 2 endocannabinoid receptors (CB2) are considered to reflect different aspects of microglia-related neuroinflammatory responses in the brain. Relative to the increase in the TSPO expression that occurs slightly later during neuroinflammation in a proinflammatory fashion, CB2 activation is considered to relate to the neuroprotective responses that occurs predominantly at an early stage of brain disorders. These findings, however, were deduced from studies with different animal samples under different experimental settings. Here, we aimed to examined the differences in TSPO binding and CB2 availability at an early stage of stroke in the same animal using positron emission tomography (PET). METHODS: We used a total of eight Sprague-Dawley rats that underwent photothrombotic stroke surgery. The binding levels of a TSPO tracer [11C](R)PK11195 and a CB2 tracer [11C]NE40 were measured at 24 h after the surgery in the same animal using PET in combination with immunohistochemistry for CB2 and several other markers. A morphological inspection was also performed with X-ray computed tomography for small animals. RESULTS: The levels of [11C]NE40 binding potential (BPND) were significantly higher in the cerebral cortical region on the lesion side than those on the non-lesion side, whereas no difference was found in the levels of [11C](R)PK11195 BPND between hemispheres. The tracer influx index (R1) data were all reduced on the lesion side irrespective of tracers. This increase in [11C]NE40 BPND was concomitant with an elevation in CB2 expression mainly within the microglia in the peri-infarct area, as shown by immunohistochemical examinations with Iba-1, CD11b/c+, and NG2+ staining. CONCLUSIONS: The present results provide in vivo evidence of different responses of microglia occurring in the acute state of stroke. The use of the CB2 tracer [11C]NE40 allows us to evaluate the roles played by the neuroprotective aspect of microglia in acute neuroinflammatory processes.

In vivo and In vitro Identification of Endocannabinoid Signaling in Periodontal Tissues and Their Potential Role in Local Pathophysiology.

The endocannabinoid system (ECS) with its binding receptors CB1 and CB2 impacts multiple pathophysiologies not only limited to neuronal psychoactivity. CB1 is assigned to cerebral neuron action, whereas CB2 is mainly expressed in different non-neuronal tissues and associated with immunosuppressive effects. Based on these tissue-selective CB receptor roles, it was the aim of this study to analyze potential expression in periodontal tissues under physiological conditions and inflammatory states. In vivo, CB receptor expression was investigated on human periodontal biopsies with or without bacterial inflammation and on rat maxillae with or without sterile inflammation. In vitro analyses were performed on human periodontal ligament (PDL) cells at rest or under mechanical strain via qRT-PCR, Western blot, and immunocytochemistry. P < 0.05 was set statistical significant. In vivo, CB1 expression was significantly higher in healthy PDL structures compared to CB2 (13.5% ± 1.3 of PDL tissues positively stained; 7.1% ± 0.9). Bacterial inflammation effected decrease in CB1 (9.7% ± 2.4), but increase in CB2 (14.7% ± 2.5). In contrast, sterile inflammation caused extensive CB1 (40% ± 1.9) and CB2 (41.7% ± 2.2) accumulations evenly distributed in the tooth surrounding PDL. In vitro, CB2 was ubiquitously expressed on gene and protein level. CB1 was constitutively expressed on transcriptional level (0.41% ± 0.09), even higher than CB2 (0.29% ± 0.06), but undetectable on protein level. Analyses further revealed expression changes of both receptors in mechanically loaded PDL cells. CB1 and CB2 are varyingly expressed in periodontal tissues, both adjusted by different entities of periodontal inflammation and by mechanical stress. This indicates potential ECS function as regulatory tool in controlling of periodontal pathophysiology.

Application of Strep-Tactin XT for affinity purification of Twin-Strep-tagged CB2, a G protein-coupled cannabinoid receptor.

Human cannabinoid receptor CB2 belongs to the class A of G protein-coupled receptor (GPCR). CB2 is predominantly expressed in membranes of cells of immune origin and is implicated in regulation of metabolic pathways of inflammation, neurodegenerative disorders and pain sensing. High resolution structural studies of CB2 require milligram quantities of purified, structurally intact protein. While we previously reported on the methodology for expression of the recombinant CB2 and its stabilization in a functional state, here we describe an efficient protocol for purification of this protein using the Twin-Strep-tag/Strep-Tactin XT system. To improve the affinity of interaction of the recombinant CB2 with the resin, the double repeat of the Strep-tag (a sequence of eight amino acids WSHPQFEK), named the Twin-Strep-tag was attached either to the N- or C-terminus of CB2 via a short linker, and the recombinant protein was expressed in cytoplasmic membranes of E. coli as a fusion with the N-terminal maltose binding protein (MBP). The CB2 was isolated at high purity from dilute solutions containing high concentrations of detergents, glycerol and salts, by capturing onto the Strep-Tactin XT resin, and was eluted from the resin under mild conditions upon addition of biotin. Surface plasmon resonance studies performed on the purified protein demonstrate the high affinity of interaction between the Twin-Strep-tag fused to the CB2 and Strep-Tactin XT with an estimated Kd in the low nanomolar range. The affinity of binding did not vary significantly in response to the position of the tag at either N- or C-termini of the fusion. The binding capacity of the resin was several-fold higher for the tag located at the N-terminus of the protein as opposed to the C-terminus- or middle of the fusion. The variation in the length of the linker between the double repeats of the Strep-tag from 6 to 12 amino acid residues did not significantly affect the binding. The novel purification protocol reported here enables efficient isolation of a recombinant GPCR expressed at low titers in host cells. This procedure is suitable for preparation of milligram quantities of stable isotope-labelled receptor for high-resolution NMR studies.

Expression of cannabinoid 1 and, 2 receptors and the effects of cannabinoid 1 and, 2 receptor agonists on detrusor overactivity associated with bladder outlet obstruction in rats.

BACKGROUND: This study investigated changes in the expression of cannabinoid (CB) receptors and the effects of CB1 and CB2 agonists on detrusor overactivity (DO) associated with bladder outlet obstruction (BOO) in rats. METHODS: Male Sprague Dawley rats were randomly assigned to four groups (n = 10) in each group. The control group comprised sham-operated rats. A animals in the BOO, CB1 agonist and CB2 agonist groups all underwent BOO surgery. Three weeks postoperatively, cystometrography (CMG) was performed on all rats. After confirming the presence of DO in the CB1 and CB2 agonist groups, a CB1 agonist (WIN 55,212-2) and a CB2 agonist (CB65) were instilled intravesically, and CMG was repeated. CMG parameters, including the contraction interval (CI) and contraction pressure (CP) were then analyzed. The bladders of rats in all four groups were excised following CMG. Immunofluorescence staining and Western blotting were performed to localize CB1 and CB2 and measure their expression levels in the urothelium and detrusor muscle. RESULTS: The CI was significantly longer and the CP was significantly lower in the CB1 agonist group than in the BOO group. CI and CP in the CB2 agonist group showed the same results. CB1 receptor immunofluorescence staining signals and immunoreactive bands in Western blotting were increased in the BOO group compared with results in the control group. Similarly, results for the CB2 receptor were also increased in the BOO group, although this difference was not significant. The CMG parameters in the BOO group were significantly improved by the inhibitory effects of CB1 and CB2 agonists on BOO-associated DO. The expression of CB1 was significantly increased in the urothelium and detrusor muscle in BOO-associated DO, but no significant change in CB2 expression was observed. CONCLUSIONS: CB1 and CB2 receptors, especially CB1, play a role in the pathophysiology of BOO-associated DO, and could serve as therapeutic targets.

Evaluation of cannabinoid CB1 and CB2 receptors expression in mobile tongue squamous cell carcinoma: associations with clinicopathological parameters and patients' survival.

Cannabinoid receptors (CB1R and CB2R) constitute essential members of the endocannabinoid system (ECS) which participates in many different functions indispensable to homeostatic regulation in several tissues, exerting also antitumorigenic effects. The present study aimed to assess the clinical significance of CB1R and CB2R protein expression in mobile tongue squamous cell carcinoma (SCC). CB1R and CB2R expression was assessed immunohistochemically on 28 mobile tongue SCC tissue samples and was analyzed in relation with clinicopathological characteristics and overall and disease-free patients' survival. CB1R, CB2R, and concomitant CB1R/CB2R expression was significantly increased in older compared to younger mobile tongue SCC patients (p = 0.0243, p = 0.0079, and p = 0.0366, respectively). Enhanced CB2R and concomitant CB1R/CB2R expression was significantly more frequently observed in female compared to male mobile tongue SCC patients (p = 0.0025 and p = 0.0016, respectively). Elevated CB2R expression was significantly more frequently observed in mobile tongue SCC patients presenting well-defined tumor shape compared to those with diffuse (p = 0.0430). Mobile tongue SCC patients presenting enhanced CB1R, CB2R, or concomitant CB1R/CB2R expression showed significantly longer overall (log-rank test, p = 0.004, p = 0.011, p = 0.018, respectively) and disease-free (log-rank test, p = 0.003, p = 0.007, p = 0.027, respectively) survival times compared to those with low expression. In multivariate analysis, CB1R was identified as an independent prognostic factor for disease-free patients' survival (Cox-regression analysis, p = 0.032). The present study provides evidence that CB1R and CB2R may play a role in the pathophysiological aspects of the mobile tongue SCC and even each molecule may constitute a potential target for the development of novel anti-cancer drugs for this type of malignancy.

Dronabinol has preferential antileukemic activity in acute lymphoblastic and myeloid leukemia with lymphoid differentiation patterns.

BACKGROUND: It has been previously demonstrated in several cancer models, that Dronabinol (THC) may have anti-tumor activity--however, controversial data exists for acute leukemia. We have anecdotal evidence that THC may have contributed to disease control in a patient with acute undifferentiated leukemia. METHODS: To test this hypothesis, we evaluated the antileukemic efficacy of THC in several leukemia cell lines and native leukemia blasts cultured ex vivo. Expression analysis for the CB1/2 receptors was performed by Western immunoblotting and flow cytometry. CB-receptor antagonists as well as a CRISPR double nickase knockdown approach were used to evaluate for receptor specificity of the observed proapoptotic effects. RESULTS: Meaningful antiproliferative as well as proapoptotic effects were demonstrated in a subset of cases--with a preference of leukemia cells from the lymphatic lineage or acute myeloid leukemia cells expressing lymphatic markers. Induction of apoptosis was mediated via CB1 as well as CB2, and expression of CB receptors was a prerequisite for therapy response in our models. Importantly, we demonstrate that antileukemic concentrations are achievable in vivo. CONCLUSION: Our study provides rigorous data to support clinical evaluation of THC as a low-toxic therapy option in a well defined subset of acute leukemia patients.

MicroRNA-665 is involved in the regulation of the expression of the cardioprotective cannabinoid receptor CB2 in patients with severe heart failure.

The myocardial endocannabinoid system has been linked to stress response and cardioprotection. In chronic heart failure (CHF), protective CB2 receptors are markedly up-regulated while CB1 receptors are slightly down-regulated. We here provide evidence that myocardial CB receptors are subject to microRNA regulation. By a combined computational and experimental approach we show that CB1 receptors are regulated by miR-494, and CB2 receptors are targeted by miR-665. Moreover, we demonstrate that in CHF, miR-665 expression is significantly decreased while miR-494 is slightly increased, which is concordant with the previously reported alterations of CB receptors. These results suggest that in CHF, altered expression of specific miRNAs may contribute to a compensatory response of the diseased myocardium.

Lactobacillus acidophilus modulates intestinal pain and induces opioid and cannabinoid receptors.

Abdominal pain is common in the general population and, in patients with irritable bowel syndrome, is attributed to visceral hypersensitivity. We found that oral administration of specific Lactobacillus strains induced the expression of mu-opioid and cannabinoid receptors in intestinal epithelial cells, and mediated analgesic functions in the gut-similar to the effects of morphine. These results suggest that the microbiology of the intestinal tract influences our visceral perception, and suggest new approaches for the treatment of abdominal pain and irritable bowel syndrome.

Intrathecal injection of JWH015 attenuates remifentanil-induced postoperative hyperalgesia by inhibiting activation of spinal glia in a rat model.

BACKGROUND: Hyperalgesia and neuroinflammation are associated with glia, which consists of macroglia and microglia. In this study, we used a selective cannabinoid receptor type 2 (CB2) agonist JWH015 to investigate remifentanil-induced postoperative hyperalgesia. METHODS: Mechanical allodynia and thermal hyperalgesia after postoperative hyperalgesia and intrathecal injection of JWH015 were assessed by the paw withdrawal mechanical threshold and paw withdrawal thermal latency tests. We used immunohistochemistry and immunoblotting to investigate the effect of JWH015 on CB2 receptor, NR2B subunits, activated glial cells, and proinflammatory cytokine expression in rats after remifentanil-induced postoperative hyperalgesia. RESULTS: Postoperative hyperalgesia was induced by intraoperative infusion of remifentanil. Glial cells were activated, and expression levels of several genes were significantly increased, including interleukin 6, tumor necrosis factor α, CB2, and the NR2B subunit phosphorylated at Tyr-1472 (p-NR2B). Intrathecal injection of JWH015 significantly inhibited glial cell activation, suppressed expression of interleukin 6, tumor necrosis factor α, and p-NR2B, and stimulated CB2 expression, thus attenuating postoperative hyperalgesia. However, these phenomena were abolished in the group that was preadministered with AM630. CONCLUSIONS: The activation of glia, the production of proinflammatory cytokines, and the expression of CB2 and p-NR2B in the spinal dorsal horn increase significantly during the process of remifentanil-induced hyperalgesia. These changes can be regulated by pretreatment with JWH015, which may be the main mechanism underlying the antihyperalgesia effects of JWH015.

Cannabinoid receptor 2 as a potential therapeutic target in rheumatoid arthritis.

BACKGROUND: Some of cannabinoids, which are chemical compounds contained in marijuana, are immunosuppressive. One of the receptors, CB receptor 1 (CB1), is expressed predominantly by the cells in the central nervous system, whereas CB receptor 2 (CB(2)) is expressed primarily by immune cells. Theoretically, selective CB(2) agonists should be devoid of psychoactive effects. In this study, we investigated therapeutic effects of a selective CB(2) agonist on arthritis. METHODS: The expression of CB(2) was analyzed with immunohistochemistry and Western blotting. Interleukin (IL)-6, matrix metalloproteinase-3 (MMP-3), and chemokine (C-C motif) ligand 2 (CCL2) were quantified with enzyme-linked immunosorbent assays (ELISA). Osteoclastogenesis was assessed with tartrate-resistant acid phosphatase staining and the resorption of coated-calcium phosphate. Effect of JWH133, a selective CB(2) agonist, on murine collagen type II (CII)-induced arthritis (CIA) was evaluated with arthritis score, and histological and radiographic changes. IFN-γ and IL-17 production by CII-stimulated splenocytes and serum anti-CII Ab were analyzed by ELISA. RESULTS: Immunohistochemistry showed that CB(2) was expressed more in the synovial tissues from the rheumatoid joints than in those from the osteoarthritis joints. CB(2) expression on RA FLS was confirmed with Western blot analysis. JWH133 inhibited IL-6, MMP-3, and CCL2 production from tumor necrosis factor-α-stimulated fibroblast-like synoviocytes (FLS) derived from the rheumatoid joints, and osteoclastogenesis of peripheral blood monocytes. Administration of JWH133 to CIA mice reduced the arthritis score, inflammatory cell infiltration, bone destruction, and anti-CII IgG1 production. CONCLUSION: The present study suggests that a selective CB(2) agonist could be a new therapy for RA that inhibits production of inflammatory mediators from FLS, and osteoclastogenesis.

Depression exacerbates myocardial ischemia-reperfusion injury in mice via CNR2 gene and MIF-AMPK signaling pathway.

BACKGROUND: Myocardial ischemia-reperfusion(I/R)injury constitute the fundamental pathophysiology of acute myocardial infarction (AMI). Ischemic heart releases macrophage migration inhibitory factor (MIF), which activates MIF- AMPK signaling pathway. Depression is a significant risk factor for AMI. In a state of depression, peripheral expression of cannabinoid receptor 2 (CNR2) genes was downregulated. AIMS: We investigated the mechanism by which depression exacerbates myocardial I/R injury through the CNR2 and MIF-AMPK signaling pathways. METHODS: We established mouse models of depression and myocardial I/R. Left ventricular function was assessed using cardiac ultrasound and TTC staining. The protein levels of myocardial CNR2, MIF, AMPK, and ACC were determined by Western blot, while the expression level of CNR2 was measured using RT-qPCR. Additionally, MIF content in peripheral blood was quantified using ELISA. RESULTS: After I/R, the expression level of CNR2 was found to be lower in the depression group, leading to a deterioration in left heart function. Depressed mice exhibited lower secretion of MIF, accompanied by a decrease in the activation of the MIF-AMPK signaling pathway. However, injection of CNR2 agonist JWH133 prior to ischemia increased the activation of the MIF-AMPK signaling pathway, while CNR2 inhibitor AM630 decreased the activation. LIMITATIONS: Further research is needed to investigate the specific neuroendocrine mechanism affecting myocardial CNR2 expression in depression. And these experimental conclusions require further verification at the cellular level. CONCLUSIONS: The activation of CNR2 in myocardium following I/R is impeded by depression, thereby exacerbating myocardial I/R injury through attenuation of the MIF-AMPK signaling pathway activation.

Cannabinoid receptors 1 and 2 are associated with bladder dysfunction in an experimental diabetic rat model.

OBJECTIVE: To investigate diabetes-associated changes in urinary bladder expression of cannabinoid receptors 1 and 2 (CB1 and CB2) and the functional role of CB agonists and antagonists in mediating phasic contractions of isolated bladder strips using a streptozotocin-induced diabetic rat model. MATERIALS AND METHODS: The bladder and dorsal root ganglion (DRG) were removed from diabetic rats and age-matched controls 8-10 weeks after diabetes induction. Expression of CB1 and CB2 mRNA was studied using quantitative real-time PCR and protein levels were determined by Western blot analysis. The effect of increasing concentrations (0.1-100 µM) of the mixed CB1/CB2 agonist R(+)-WIN 55,212-2 (WIN), selective CB1 antagonist (AM251) and selective CB2 antagonist (AM630) on carbachol-evoked contraction of bladder strips from control and diabetic rats was investigated. WIN-induced alterations of bladder strip contraction were then studied after pre-incubation with AM251 and AM630. RESULTS: Diabetes induced decreased CB1 protein and mRNA expression in both the bladder and DRG (P < 0.05), while decreased CB2 expression was observed in the bladder (P < 0.05). WIN decreased the amplitude, but not frequency, of carbachol-induced phasic contractions of bladder strips in a concentration-dependent manner and this effect was diminished in the diabetic state. AM630 and AM251 had no effect on isolated detrusor muscle function. Moreover, pre-incubation with AM251 partially counteracted the effect of WIN on detrusor muscle contraction. CONCLUSION: The results indicate that CB1 and CB2 are responsible for the pathogenesis of bladder dysfunction in diabetes mellitus and represent a viable target for pharmacological treatment of bladder cystopathy.

Temporal and behavioral variability in cannabinoid receptor expression in outbred mice submitted to ethanol-induced locomotor sensitization paradigm.

BACKGROUND: There is a close relationship between the endocannabinoid system and alcoholism. This study investigated possible differential expression of cannabinoid receptors CB1 (CB1R) and CB2 (CB2R) in an outbred mice strain displaying behavioral variability to ethanol (EtOH)-induced locomotor sensitization. METHODS: Male adult Swiss mice treated chronically with EtOH (2 g/kg, i.p., daily for 21 days) were classified as "EtOH_High" or "EtOH_Low" according to their locomotor activity after the 21st EtOH injection. A control group was similarly injected with saline. Temporal analysis of CB1R and CB2R immunoreactivity was performed in 3 different occasions: (i) at the end of chronic EtOH treatment, (ii) on the fifth day of EtOH withdrawal, and (iii) after EtOH challenge. RESULTS: Overall, no differences were seen between experimental groups regarding the CB1R at the end of acquisition. However, there were decreases in CB2R in the prefrontal cortex and the hippocampus in EtOH_Low mice. On the fifth day of withdrawal, only EtOH_High mice presented increase in CB1R. Nonetheless, CB2R up-regulation was observed in both EtOH_High and EtOH_Low mice. EtOH challenge counteracted CB1R and CBR2 up-regulation, mainly in the EtOH_High, in structures related to emotionality, such as prefrontal cortex, ventral tegmental area, amygdala, striatum, and hippocampus. CONCLUSIONS: There are different patterns of cannabinoid receptor expression during locomotor sensitization paradigm, at both temporal and behavioral perspectives. We hypothesize that CB2R down-regulation might be related to resilience to develop locomotor sensitization, while CB1R up-regulation relates to withdrawal aspects in sensitized mice.

CB1 and CB2 receptor expression and promoter methylation in patients with cannabis dependence.

CB1 and CB2 receptors are influenced via exogenous and endogenous cannabinoids. To date, little is known regarding changes in receptor expression and methylation in THC (tetrahydrocannabinol) dependence. Therefore, the CB1 and CB2 receptor mRNA expression levels and promoter methylation status in the peripheral blood cells of 77 subjects (36 with THC dependence, 21 cigarette smokers and 20 nonsmokers) were assessed by quantitative real-time PCR and methylation-specific PCR. There was a significant difference in CB1 receptor expression levels between the three groups (ANOVA, p < 0.001, d.f. = 2, F = 71.3). The mean promoter methylation (%) was significantly negatively correlated with CB1 receptor mRNA expression levels (Spearman's rho: r = -0.37; p = 0.002). Using a mixed general linear model, it was demonstrated that the CB1 mRNA expression (as the dependent variable) was associated with the satisfaction with life scale (SWLS) (r = 0.101; T = 2.8; p = 0.007), craving (as measured with the VAS; r = -0.023; T = -2.3; p = 0.023) and the WHO-Assist Subscale for Cannabis consumption (r = -0.068; T = -2.4; p = 0.02). CB1 receptor expression levels and methylation status appear to be altered in subjects with THC dependence.

Cannabinoid receptor CB2 protects against balloon-induced neointima formation.

Cannabinoid receptor CB(2) activation inhibits inflammatory proliferation and migration of vascular smooth muscle cells in vitro. The potential in vivo relevance of these findings is unclear. We performed carotid balloon distension injury in hypercholesterolemic apolipoprotein E knockout (ApoE(-/-)) mice receiving daily intraperitoneal injection of the CB(2) agonist JWH133 (5 mg/kg) or vehicle, with the first injection given 30 min before injury. Alternatively, we subjected CB(2)(-/-) and wild-type (WT) mice to balloon injury. We determined CB(2) mRNA and protein expression in dilated arteries of ApoE(-/-) mice. Neointima formation was assessed histologically. We used bone marrow-derived murine CB(2)(-/-) and WT macrophages to study adhesion to plastic, fibronectin, or collagen, and migration was assayed by modified Boyden chamber. Aortic smooth muscle cells were isolated to determine in vitro proliferation rates. We found increased vascular CB(2) expression in ApoE(-/-) mice in response to balloon injury. Seven to twenty-one days after dilatation, injured vessels of JWH133-treated mice had less intimal nuclei numbers as well as intimal and medial areas, associated with less staining for proliferating cells, smooth muscle cells, and macrophages. Complete endothelial repair was observed after 14 days in both JWH133- and vehicle-treated mice. CB(2) deficiency resulted in increased intima formation compared with WT, whereas JWH133 did not affect intimal formation in CB(2)(-/-) mice. Apoptosis rates assessed by in situ terminal deoxynucleotidyl transferase dUTP-mediated nick-end labeling staining 1 h postballooning were significantly higher in the CB(2) knockouts. In vitro, bone marrow-derived CB(2)(-/-) macrophages showed enhanced adherence and migration compared with WT cells and elevated mRNA levels of adhesion molecules, chemokine receptors CCR1 and 5, and chemokine CCL2. Proliferation rates were significantly increased in CB(2)(-/-) smooth muscle cells compared with WT. In conclusion, pharmacological activation or genetic deletion of CB(2) receptors modulate neointima formation via protective effects in macrophages and smooth muscle cells.

Mistic and TarCF as fusion protein partners for functional expression of the cannabinoid receptor 2 in Escherichia coli.

G protein coupled receptors (GPCRs) are key players in signal recognition and cellular communication making them important therapeutic targets. Large-scale production of these membrane proteins in their native form is crucial for understanding their mechanism of action and target-based drug design. Here we report the overexpression system for a GPCR, the cannabinoid receptor subtype 2 (CB2), in Escherichia coli C43(DE3) facilitated by two fusion partners: Mistic, an integral membrane protein expression enhancer at the N-terminal, and TarCF, a C-terminal fragment of the bacterial chemosensory transducer Tar at the C-terminal of the CB2 open reading frame region. Multiple histidine tags were added on both ends of the fusion protein to facilitate purification. Using individual and combined fusion partners, we found that CB2 fusion protein expression was maximized only when both partners were used. Variable growth and induction conditions were conducted to determine and optimize protein expression. More importantly, this fusion protein Mistic-CB2-TarCF can localize into the E. coli membrane and exhibit functional binding activities with known CB2 ligands including CP55,940, WIN55,212-2 and SR144,528. These results indicate that this novel expression and purification system provides us with a promising strategy for the preparation of biologically active GPCRs, as well as general application for the preparation of membrane-bound proteins using the two new fusion partners described.

Prevention of paclitaxel-induced neuropathy through activation of the central cannabinoid type 2 receptor system.

BACKGROUND: Peripheral neuropathy is a major dose-limiting toxicity of chemotherapy, especially after multiple courses of paclitaxel. The development of paclitaxel-induced neuropathy is associated with the activation of microglia followed by the activation and proliferation of astrocytes, and the expression and release of proinflammatory cytokines in the spinal dorsal horn. Cannabinoid type 2 (CB(2)) receptors are expressed in the microglia in neurodegenerative disease models. METHODS: To explore the potential of CB(2) agonists for preventing paclitaxel-induced neuropathy, we designed and synthesized a novel CB(2)-selective agonist, namely, MDA7. The effect of MDA7 in preventing paclitaxel-induced allodynia was assessed in rats and in CB(2)(+/+) and CB(2)(-/-) mice. We hypothesized that the CB(2) receptor functions in a negative-feedback loop and that early MDA7 administration can blunt the neuroinflammatory response to paclitaxel and prevent mechanical allodynia through interference with specific signaling pathways. RESULTS: We found that MDA7 prevents paclitaxel-induced mechanical allodynia in rats and mice in a dose- and time-dependent manner without compromising paclitaxel's antineoplastic effect. MDA7's neuroprotective effect was absent in CB(2)(-/-) mice and was blocked by CB(2) antagonists, suggesting that MDA7's action directly involves CB(2) receptor activation. MDA7 treatment was found to interfere with early events in the paclitaxel-induced neuroinflammatory response as evidenced by relatively reduced toll-like receptor and CB(2) expression in the lumbar spinal cord, reduced levels of extracellular signal-regulated kinase 1/2 activity, reduced numbers of activated microglia and astrocytes, and reduced secretion of proinflammatory mediators in vivo and in in vitro models. CONCLUSIONS: Our findings suggest an innovative therapeutic approach to prevent chemotherapy-induced neuropathy and may permit more aggressive use of active chemotherapeutic regimens with reduced long-term sequelae.